High-SPEED super-resolution SPEED microscopy to study primary cilium signaling in vivo.

The primary cilium is a surface exposed organelle found in eukaryotic cells that functions to decode a variety of intracellular signals with significant implications in human developmental disorders and diseases. It is therefore highly desirable to obtain in vivo information regarding the dynamic processes occurring within the primary cilium. However, current techniques are limited by either the physical limitations of light microscopy or the static nature of electron microscopy. To overcome these limitations, single-point edge-excitation sub-diffraction (SPEED) microscopy was developed to obtain dynamic in vivo information in subcellular organelles such as cilia and nuclear pore complexes using single-molecule super-resolution light microscopy with a spatiotemporal resolution of 10-20nm and 0.4-2ms. Three-dimensional (3D) structural and dynamic information in these organelles can be further obtained through a post-processing 2D-to-3D transformation algorithm. Here we present a modular step-by-step protocol for studying primary cilium signaling dynamics, including Intraflagellar transport (IFT) via IFT20 and somatostatin g-protein-coupled receptor activity via SSTR3.